Prior research has shown that neurons within the spinal cord are sensitive to response-outcome (instrumental) relationships. Rats with complete spinal cord transections can learn to maintain the hindlimb in a flexed position if leg shock is delivered when the leg is extended (response- contingent shock). Using this simple preparation, we have shown that stimulation alters the capacity for spinal learning in a bidirectional manner. Training with response-contingent shock promotes later spinal learning. Conversely, nociceptive stimulation that is independent of leg position (uncontrollable shock or peripheral paw injury) inhibits future spinal learning and impairs locomotor recovery after contusive spinal cord injury (SCI). Prior work has found that these impairments in spinal learning depend on a maladaptive form of glutamate-mediated plasticity that impairs future use-dependent plasticity in the spinal cord. The cellular mechanisms regulating this plasticity of plasticity ("metaplasticity") are not well-understood. Our hypothesis is that the cytokine tumor necrosis factor a (TNFa) within the spinal cord plays a critical mechanistic role in spinal learning impairments after uncontrollable stimulation. TNFa is released in elevated levels after SCI or nociceptive stimulation. TNFa has recently been found to alter synaptic plasticity within the injured spinal cord by increasing trafficking of the glutamate AMPA receptor (AMPAR) to the plasma membrane of spinal neurons. Preliminary data suggest that TNFa-induced AMPAR trafficking may contribute to spinal learning impairments after SCI. Intrathecal delivery of an AMPAR agonist or TNFa impairs spinal learning. Conversely a TNFa inhibitor promotes spinal learning. Aim 1 establishes the dose-response and temporal features of these TNFa-mediated effects. Aim 2 evaluates TNFa mRNA and protein levels in the spinal cord after uncontrollable stimulation using qRT-PCR, ELISA, in situ hybridization and immunofluorescence. Aim 3 examines TNF-induced trafficking of AMPARs to the plasma membrane of spinal neurons after uncontrollable stimulation by biochemical and confocal microscopy methods. Aim 4 tests the therapeutic potential of a TNFa1 inhibitor for promoting use-dependent plasticity and recovery of function after contusive SCI. Our long-term goal is to unravel the mechanisms that regulate adaptive spinal plasticity, allowing patients to re-establish essential functions, while limiting the maladaptive plasticity that can lead to spasticity or intractable pain. By defining key mechanisms that disable spinal cord learning and recovery of function, we hope to provide novel therapeutic targets that promote spinal cord learning and neurorehabilitation after SCI. PUBLIC HEALTH RELEVANCE: Project Narrative/Public Health Relevance Statement Spinal cord Injury (SCI) produces a devastating syndrome that is characterized by loss of motor control and mobility, as well as sensory dysfunction and pain. The proposed project explores cellular mechanisms that regulate a form of spinal cord learning that is thought to contribute to recovery of function after SCI. These studies may provide a novel target for improving recovery after SCI.